Project summary Neuronal activity can be modulated by transmitters that act through receptors coupled to heterotrimeric G proteins. Activation of these G protein signal transduction pathways leads to changes in neuronal excitability or synaptic transmission at the cellular level, and changes in behavior or memory at the organismal level. The long-term goal of our work is to identify and understand the mechanistic basis of neuromodulatory pathways. The goal of this proposal is to identify new regulatory pathways that lead to the modulation of a specific ion channel, the NALCN/NCA channel. The NALCN/NCA ion channel is a putative cation channel related to voltage-gated sodium and calcium channels, but whose precise cellular role and regulation are not well understood. However, mutations in NALCN or its associated subunits have been directly linked to human neurological diseases characterized by a range of symptoms, including abnormal movements and muscle contractions, intellectual disability, and seizures. Additionally, mutations in this channel in model organisms lead to strong neuronal phenotypes including defects in rhythmic behaviors, neuronal excitability, and synaptic function, demonstrating the physiological importance of this channel. Through a forward genetic screen in the nematode C. elegans, we found that the NCA ion channel is activated by a new signal transduction pathway acting downstream of the heterotrimeric G protein Gq. Activated Gq directly binds and stimulates the guanine nucleotide exchange activity of the Trio RhoGEF to activate the small G protein Rho. Here we will determine how this Gq-Rho pathway activates NCA by studying additional factors identified in our screen. In Aim 1, we will focus on a G protein-coupled receptor kinase. Our hypothesis is that this kinase modulates dopamine signaling that specifically regulates NCA activity through the Gq-Rho pathway. We will perform a structure- function analysis of this kinase to determine its functional domains and perform genetic and biochemical experiments to determine how this kinase interacts with dopamine receptors and members of the Rho-Nca pathway. In Aim 2, we will focus on a mitogen-activated protein kinase (MAPK) pathway that modulates Gq- Rho activation of NCA. We will identify the members of this signaling pathway and determine how they modulate output of the Gq-Rho-Nca pathway. The proposed work is significant because it will identify the signaling pathways that modulate neuronal and synaptic activity via a physiologically and medically important ion channel. The proposed work is innovative because it will close gaps in our understanding of how the NALCN/NCA ion channel is activated and identify new mechanisms of regulation of this channel.